TOXOPLASMA GONDII IN WILD RUMINANTS BRED IN GAME PRESERVES AND FARMS WITH PRODUCTION DESTINED FOR HUMAN CONSUMPTION IN THE CZECH REPUBLIC

Toxoplasma gondii is the causative agent of the most common parasitic infection in humans. Almost all warm-blooded animals, as well as humans, can act as intermediate hosts that harbour infective cysts in their tissues. Felids act as definitive hosts excreting oocysts in faeces. In humans, T. gondii can cause subclinical infection but also severe clinical disease with a wide range of symptoms, especially in immunocompromised individuals. The infection is usually asymptomatic in animals and is not recognized at either anteor post-mortem inspection. The consumption of undercooked meat from infected animals is one of the most important routes by which the infection can be transmitted to humans. Handling of the organs and other tissues of game animals and eating their undercooked meat have been described as a risk of T. gondii infection. For diagnosis of toxoplasmosis, the combination of serological and molecular methods has been described as a suitable approach. Antibodies against T. gondii were detected in 20.8%, 50.0%, 23.1%, and 24.4% of red deer, sika deer, fallow deer and mouflons, respectively, coming from game preserves and farms in the Czech Republic. T. gondii DNA was found in the muscle tissue of red deer (8.3%) and mouflons (14.6%). The lower prevalence rates based on molecular screening could be due to the random distribution and low density of cysts in tissues of infected animals. Bearing in mind the increase in the number of hunted animals and the growing trend in game consumption, it is important to educate hunters and game meat consumers about the risk of exposure to this zoonotic infection during handling and consumption of the


INTRODUCTION
Toxoplasma gondii is a ubiquitous zoonotic parasite of significant concern to human health (EFSA, 2007).Toxoplasma only rarely causes severe clinical symptoms, the infection is mostly asymptomatic or only mild symptoms occur (self-limiting lymphadenopathy, fever or intraocular inflammation).However, it can cause life-threatening infections in immunocompromised individuals (disseminated disease with encephalitis, meningoencephalitis, myocarditis or hepatitis).Parasitaemia in a primarily infected pregnant woman may result in congenital toxoplasmosis with abortion, neonatal death, or fetal damage as encephalomyelitis, retinochoroiditis, intracranial calcifications, hydrocephalus or mental retardation in survivors (Tenter et al., 2000;EFSA, 2007;Kijlstra and Jongert, 2008).
Almost all warm-blooded animals, as well as humans, can serve as intermediate hosts of Toxoplasma with the formation of infective tissue cysts having a high affinity for neural and muscular tissues.Tissue cysts may persist for the life of the hosts.Wild and domestic cats and other felids act as definitive hosts excreting oocysts in their faeces.Infection in hosts, including humans, can be acquired by the consumption of raw or undercooked meat containing tissue cysts, from soil, water or food contaminated by oocysts, or congenitally.Also, contact with infected carcasses during evisceration, dressing and processing presents a risk of infection to humans The infection usually does not cause clinical signs in animals or visible lesions in carcasses and is not recognized at either ante-or post-mortem inspection.Although toxoplasmosis is the most reported parasitic zoonosis in humans in EU, there is inadequate system for routine monitoring and therefore the incidence of toxoplasmosis in humans and animals and the presence of T. gondii in food is underestimate (EFSA, 2007).According to the European Food Safety Authority (EFSA), T. gondii was identified as a relevant biological hazard to be addressed in revised meat inspection regulations for pigs, sheep, goats, farmed deer and farmed wild boar (EFSA, 2011; 2013a; 2013b In humans, T. gondii can cause subclinical infection but also severe clinical disease with a wide range of symptoms, especially in immunocompromised individuals.The infection is usually asymptomatic in animals and is not recognized at either ante-or post-mortem inspection.The consumption of undercooked meat from infected animals is one of the most important routes by which the infection can be transmitted to humans.Handling of the organs and other tissues of game animals and eating their undercooked meat have been described as a risk of T. gondii infection.For diagnosis of toxoplasmosis, the combination of serological and molecular methods has been described as a suitable approach.Antibodies against T. gondii were detected in 20.8%, 50.0%, 23.1%, and 24.4% of red deer, sika deer, fallow deer and mouflons, respectively, coming from game preserves and farms in the Czech Republic.T. gondii DNA was found in the muscle tissue of red deer (8.3%) and mouflons (14.6%).The lower prevalence rates based on molecular screening could be due to the random distribution and low density of cysts in tissues of infected animals.Bearing in mind the increase in the number of hunted animals and the growing trend in game consumption, it is important to educate hunters and game meat consumers about the risk of exposure to this zoonotic infection during handling and consumption of the meat.
Volume 9 289 No. 1/2015 Molecular methods, which can detect the genome of the parasite in tissues but without confirmation of its viability, are less sensitive because the density of these parasites is low in meat (Dubey, 1988;Dubey et al., 2014).
According to EFSA, there are limited data on the prevalence of toxoplasmosis predominantly in farmed cervids (EFSA, 2013b).By means of both serological and PCR testing, the aim of this study was to determine the prevalence of T. gondii in wild ruminants from game preserves and farms in the Czech Republic, destined for human consumption.

MATERIAL AND METHODOLOGY
During the period 2012-2014, muscle samples (musculus gluteus, diaphragma and/or masseter) were collected from 82 carcasses of wild ruminants (red deer, sika deer, fallow deer, and mouflons) immediately following hunting and killing.Animals came from game preserves (n=5) and farms (n=6) in the Czech Republic (regions Kralovehradecky, Pardubicky, Stredocesky and Vysocina) and their meat was intended for human consumption.The ages of the majority of the animals were not available for the purposes of this study.
Meat juices were obtained by subsequent freezing and thawing of the muscle tissue and stored at -20 °C until tested for antibodies to T. gondii by the ID Screen® Toxoplasmosis Indirect Multi-species ELISA kit (IDVET, Montpellier, France) according to the manufacturer's instructions.This ELISA kit was evaluated as a screening test for toxoplasmosis in sera or meat juice of ruminants, cats, dogs and pigs and was also used in serological surveys in wild ruminants (Roqueplo et al., 2011).
In addition, 25 g of muscle tissue were analyzed with real time PCR specific for T. gondii.Three cases of acute toxoplasmosis in deer hunters were reported by Sacks et al. (1983).Ingestion of raw or rare venison was considered to be the most likely route of infection.
The consumption of game in the Czech Republic is relatively low and traditionally confined to the hunting fraternity.However, there is an increasing trend towards a wider appreciation amongst those who seek occasional dietary diversification (0.5 kg of game per capita per year in 2006 in comparison with 0.9 kg in 2013; Anonymous, 2015a).This increase is linked to the growth of wild populations and number of hunted animals in recent years (Anonymous, 2015b).We collected muscle samples from a total of 82 wild ruminants of four species whose meat was intended for human consumption.T. gondii antibodies were detected in 24.4% of animals (Table 1).Using real time PCR, the presence of T. gondii DNA was detected in tissues of eight (9.8%) animals.The lower prevalence rates, based on molecular screening as compared with serological testing, could be due to the random distribution and low density of cysts in tissues of chronically infected and asymptomatic animals (one cyst per 50-100 g of tissue; We did not find significant differences in seroprevalence amongst the investigated species.The highest prevalence of T. gondii antibodies was observed in sika deer (50.0%), but the number of available samples was too low (only two animals, Table 1).T. gondii DNA was found in tissues of red deer and a higher prevalence in mouflons (8.3% and 14.6%, respectively).Feeding habits are different in these species -deer feed mainly on grass, leaves and berries, but also on young shoots and twigs, whereas mouflons graze on short grasses and thus may be more frequently infected by oocysts shed by felids.However, the higher prevalence of infection in mouflons than in deer in our study was not confirmed by other authors (Hejlíček et al., 1997; Gauss et al., 2006;Bartova et al., 2007).
In the study of Hejlíček et al. (1997) carried out in the Czech Republic (south Bohemia) during the period 1981-1990 which targeted mainly free-ranging wild ruminants, antibodies were detected in 15% (46/303), 100% (3/3) and 10% (2/20) of red deer, fallow deer and mouflons, respectively, but tissue cysts were not isolated from these animals.However, we found a higher occurrence of antibodies, especially in red deer (20.8%) and mouflons (24.4%).It could be influenced by the origin of the investigated animals in our study coming from game preserves and farms.In these smaller, fenced areas with more cats having access and with higher animal densities, the risk of infection increases.The association between positive ELISA results for T. gondii and high animal density with the presence of domestic cats being the main sources of toxoplasmosis in wild animals, has already been described (Hejlíček et al

CONCLUSION
Although toxoplasmosis is the most commonly reported parasitic zoonosis in humans, the pathogen is not routinely monitored in food-producing animals.It is obvious from our study, and previous ones, that wild cervids and mouflons are commonly exposed to T. gondii and may serve as a reservoir of infection for humans.When game animals are killed and butchered in the field, discarded viscera left unattended can infect other animals, especially carnivores and omnivores.
To prevent the transmission of infection by ingestion, meat should be cooked well at a minimum temperature of 70 °C.It is also common to freeze game meat for later culinary use.Freezing at a temperature of at least -12 °C for 3 days should be sufficient to inactivate the parasites (Dubey et al., 1988;Tenter et al., 2000;Kijlstra and Jongert, 2008).Hunters and consumers of game should be educated about the risk of exposure to this zoonotic disease during evisceration, handling and consumption.
Tissue samples were processed according to Opsteeght et al. (2010) prior to DNA isolation based on the manufacturer's protocol (QIAGEN) slightly modified to include mechanical homogenization with zirconia/silica beads (0.2 mm) in a MagNALyser instrument (Roche, Mannheim, Germany).The isolated DNA was used as a template for the triplex real time PCR assays.The detection of T. gondii via primers and probes specific for B1 and 529rep was adopted from Lass et al. (2012) and Opsteeght et al. (2010).The previously published internal amplification control was introduced to eliminate false negative samples (Slana et al., 2008).RESULTS AND DISCUSSIONIt has been suggested that the handling of the organs and other tissues of game animals and eating their undercooked meat carries a risk to humans of potential T. gondii infection (Cook et al., 2000; EFSA, 2007).Ross et al. (2001) described acquired ocular toxoplasmosis with flulike symptoms in deer hunters after ingestion of venison.

Ross et al., 2001; EFSA, 2013b).
). Infected game can serve as a source of infection for other animals, especially carnivores, and humans (

gondii oocysts (Olamendi-Portugal et al., 2012; Ferroglio et al., 2014).
One of the suitable diagnostic methods is the detection of specific antibodies against T. gondii in serum or meat juice and seropositivity has been correlated with the presence of cysts in muscles and other animal tissues (

Alena Lorencova, Jiri Lamka, Michal Slany ABSTRACT Toxoplasma
gondii is the causative agent of the most common parasitic infection in humans.Almost all warm-blooded animals, as well as humans, can act as intermediate hosts that harbour infective cysts in their tissues.Felids act as definitive hosts excreting oocysts in faeces.

Dubey et al., 1988; Dubey et al., 2014).
Nevertheless, real time PCR is a useful method for demonstrating the presence of parasites in muscle samples of food-producing animals.T. gondii DNA was detected in four out of eight cases in

Table 1
Prevalence of Toxoplasma gondii infection in wild ruminants from game preserves and farms in the Czech Republic.

et al. (2010) detected
T. gondii antibodies in 23%, 17% and 25% of mouflons, red deer and fallow deer, respectively, and viable parasites were recovered by bioassay from one mouflon and one red deer.The identified prevalence of toxoplasmosis in deer hunted in Poland was 8.8% using direct agglutination and 11.6% using PCR, according to the report of EFSA and ECDC (EFSA, 2014).However, it is difficult to compare results from various studies because different diagnostic methods (serological and molecular) were used and the animal´s age, geographical location and climatic conditions can also influence the prevalence of infection (